Diphtheria toxin (DTx) is a two-component exotoxin of Corynebacterium diphtheriae synthesized as a single polypeptide chain of 535 amino acids containing an A (active) domain and a B (binding) domain linked together by a disulfide bridge. The toxin binds to a cell receptor (HB-EGF receptor) and enters the cell by endocytosis where the A domain is released from the B domain by proteolytic cleavage. The A domain then exits the endosome through pores made by the B domain and enters the cytoplasm where it inhibits protein synthesis ultimately resulting in cell death.
CRM197 is a mutated form of Dtx containing a single amino acid substitution of glutamic acid for glycine (G52E) that renders the protein enzymatically inactive and non-toxic. CRM197 has been found to be an ideal carrier for conjugate vaccines against encapsulated bacteria. Conjugate vaccines comprise CRM197 covalently linked to poorly immunogenic and T-cell independent capsular polysaccharides, thus creating conjugate antigens that are highly immunogenic and result in long-lasting immunity against the antigen(s).
Vaccines containing CRM197 as a carrier protein have been successfully used to immunize millions of children and include Menveo®, a tetravalent conjugate vaccine against serogroups A-C-W135-Y of Neisseria meningitidis, Menjugate® and Meningitec® (against serotype C of N. meningitidis), Vaxem-Hib® and HibTITER® (against Haemophilus influenzae type B, Hib), and the multivalent pneumococcal conjugate Prevnar™.
In contrast to tetanus and diphtheria toxins, CRM197 does not require chemical detoxification and can therefore be purified to homogeneity and used directly for conjugation. CRM197 is currently manufactured by the fermentation of either Corynebacterium diphtheriae C7, where it is expressed from multiple lysogens of the β phage, or from a plasmid system in Pseudomonas flurorescens. The yield of CRM197 (which is released into the media during C. diphtheriae fermentation) is low ranging from tens of mg/L to ˜200 mg/L and requires biosafety level 2 facilities, resulting in a retail price of about $500 US per milligram of CRM197. A single dose of vaccine typically contains about 10 and 60 μg of CRM197 and over 150 million doses are used each year. Current demand for conjugate CRM197 vaccines has outpaced supply and has resulted in delays in initiating vaccination programs in developing countries placing the health of millions of children at risk.
Moreover, a possible therapeutic use for CRM197 in treating cancers such as ovarian cancer has recently been reported, based on CRM197's ability to bind the soluble form of heparin-binding epidermal growth factor (pro-HB-EGF), which is highly expressed in some cancers. The research and development of this therapeutic potential places even more of a strain on current production methods.
The single greatest factor contributing to the high price and short supply of CRM197 is the historical inability to generate high amounts of CRM197 in the production workhorse E. coli. Although an insoluble form of CRM197 can be fermented in E. coli to relatively moderate yields, only a fraction of the insoluble product can be converted to the soluble form (Stefan et al., 2011). Producing high amounts of soluble CRM197 in E. coli has been even more challenging. A method for reliably and inexpensively producing high amounts of CRM197 for therapeutic use would constitute a significant advance in the art.